The Effection Of Transposons And Plasmid On Bla_KPC-2 Horizontal Spread In Enterobacteriaceae Bacteria

Enterobacteriaceae are most important opportunistic pathogens in hospital infections,because of the non-standard use of antimicrobial drugs, Enterobacteriaceae bacteria antimicrobial resistance condition worsing, for most of the clinical commonly used drugs the resistant rate> 50%. Carbapenems is only a few antibiotics that e. coli and klebsiella pneumonia bacteria that remains highly sensitive. Recently, there are more and more carbapenems resistant Enierobacteriaceae bacteria, drawed the experts and clinical microbiology workers attention. It is a serious threat to the anti-infection treatment. KPC (Klebsiella pneumoniae carbapenemase, KPC) is the main cause of Enterobacteriaceae bacteria carbapenems resistance. Reports suggest that transposon carry blaKPC-2 widely exists in different size, different structure of plasmid, most of the plasmid can be successfully transferred to the recipient bacterium, through conjugate test, host bacteria carry blaKPC-2 varied also, and the prevalence of KPC (coded by blaKPC-2) involves transposons, plasmid and host bacteria in China. We think the prevalence of blaKPC-2 is not only due to a simple cloning spreading, but also the results of horizontal gene transfer mediated by high activity transposon, wide host, and stability conjugative plasmid. We plan to type the transposons, plasmids and bacterias host in those Enierobacteriaceaes isolates collected from the first blaKPC-2 positive isolates to the outbreak of blaKPC-2 firstly, then further study the characteristics and mechanism of the these three key roles, so that we comprehensively portray the dissemination mechanism of blaKPC-2 to provide a reliable theory for hospital infection controlling.Has great significance on monitoring of hospital infection and treatment.Part One Contribution of β-Iactamases and porin proteins OmpK35 and OmpK36 to carbapenem resistance in clinicalisolates of KPC-2-producing Klebsiella pneumoniaeObjective:To investigate the contribution of β-lactamases and porins OmpK35 and OmpK36 to carbapenem resistance in clinical isolates of KPC-2-producing Klebsiella pneumoniae.Methods:Fifty-seven consecutive, nonduplicate, carbapenem-resistant K. Pneumoniae isolates were collected and subjected to (3-lactamase-directed isoelectric focusing, PCR, gene sequencing, and MLST genotype analyses. Six representative isolates and a matching set of five blaKPC-2-minus derivatives generated by λ-Red recombination were selected for MIC testing, SDS-PAGE-based outer membrane protein analysis, and reverse transcriptase-quantitative PCR -facilitated gene expression studies.Results:Four MLST genotypes were identified among the 57 isolates; 50 belonged to ST11,5 to ST423,1 to ST65, and 1 to a novel MLST type, ST977. All six selected isolates produced β-lactamases KPC-2, TEM-1, and CTX-M-14. Expression of ompK35 and ompK36 was markedly attenuated in isolate XJ-1. Importantly, the novel variant ompK36, found in XJ-3 and XJ-6, may be functionally perturbed as it carries an inserted aspartic acid-glycine pair immediately distal to the highly conserved PEFXG motif. When blaKPC-2 was deleted from isolates XJ-1 and XJ-4, the mutants showed a>8-fold decrease in the MIC values of imipenem, meropenem, and ertapenem. In contrast, deletion of blaKPC-2 from the blaDHA-1-bearing XJ-3 and blaVIM-1-bearing XJ-5 isolates had a significantly reduced impact on the resulting levels of carbapenem resistance.Conclusions:Among the 57 carbapenem-resistant K. pneumoniae clinical isolates we studied, blaKPC-2, blaDHA-1, and/or blaVIM-1 appear to be the key factors contributing to high-level carbapenem resistance. Deficient expression of OmpK35 and/or OmpK36 seems to only serve as a cooperative factor in this process.Part Two The effection of transposons and plasmid on blaKPC-2 horizontal spread in Enterobacteriaceae bacteriaObjectives:To characterize the Prevalence and genetic environment of 108 KPC-Carbapenemase-Produeing Enierobacteriaceae isolates from HuanShan hospital in easteM China.Methods:108 KPC-carbapenemase-Produeing Enterobaeteriaceae were obtained from in Patients of HuaShan hospitals. Do drug sensitive test according to NCCLS recommended K-B paper diffusion method. Carbapenemase genes were detected by PCR. A PCR mapping approach was carried out to compare the relaxase gene and genetic context of the blaKPC-2. MLST was used to investigate the clonality of blaKPC-2 positive isolates.Conjugation,plasmid extraction and Southern hybridizations were used to clarify the resistance genes located on plasmid.The complete sequence of the resistance plasmid was determined using a whole-genome shotgun approach.And the bioinformatics methods were applied in genetic srructure analysis.Results:All strains were penicillium carbon alkene antibiotic resistance, MLST type showed 108 strains isolated strains can be divided into four type, blaKPC-2 gene environment study found that its on different transposons, transposons was located on the plasmid that can transfer through joint action, plasmid that carried blaKPC-2 in the first few isolations was type P31, plasmid caused blaKPC-2 spreading was type F12.Conclusions:The prevalence of blaKPC-2-positive resistant isolates has been increased in eastern China.The KPC-2 carbapenemase gene located on an Tn1721-IS26 complex transposon, can transfer through different types conjugatable plasmids, well reveals the level of domestic blaKPC-2 gene spread route of transmission.Part Three Transposon Tn1721 co-harboring plasmid-mediated fosfomycin resistance geen.fosA3 and cabarpenemase blaKPC-2In order to understand the genetic background and dissemination mechanism of carbrpenem resistance and fosfomycin resistance in Enierobacteriaceae isolates, a clinical E.coli isolate HS102707 and an Enteobacter aerogenes isolate HS112625 which were resistant to fosfomycin and intermediate to imipenem, and positive in both blaKPC-2 and fosA3 were studied. Beside, a clinical Klebsiella pneumonia isolate HS092839 which was resistant to carbapenem but susceptible to fosfomycin was also studied. A 70kb plasmid was successfully transferred to recipient E.coli J53 by conjugation test. PCR and southern blot showed blaKPC-2 was located on this plasmid. The complete sequence of pHS 102707 showed the plasmid has a replication gene"trfA", several plasmid stable genes, eg,parA and parB, a toxin-antitoxin system protein genes higA and higB and anti-restriction protein gene klcA and klcB, two sets of IV secretion system. A Tn1727-Tn3-like composite transposon harbored blaKPC-2 was inserted in the plasmid stable module at the 3’end of trfA. A composite IS26 transposon harbored fosA3 was inserted in Tn1721-tnpA gene in pHS102707and pHS 112625, led to the disruption of Tn1721-tnpA and the deletion of Tn1721-tnpR. However, only an IS26 with a truncated Tn21-tnpR was inserted in the same position in pHS092839. This is the first report of fosA3 and blaKPC-2 co-located in the same transposon, and the plasmid harbored this transposon is likely high stable and has a high ability of transconjugation. These findings suggest an intensive monitor is need on this type dissemination blaKPC-2 and fosA3.